Clathrin-mediated endocytosis (CME) is the major pathway for selective uptake of surface receptors and their bound ligands into cells. As such, CME controls many aspects of cellular homeostasis, including nutrient uptake, the surface expression of ion, sugar and other metabolite transporters, interactions with the immune system, regulation of signaling receptors, regulation of cell adhesion receptors, etc. At the synapse, CME is the major pathway for synaptic vesicle recycling and thus for maintaining neurotransmission. Most, if not all, of the component parts of the clathrin- mediated endocytotic machinery have been identified but the exact of function of few of these are known. The best studied components are the coat proteins, clathrin and adaptor proteins, and the GTPase dynamin. Many of the other parts were identified based on their interactions with these major constituents. We have developed quantitative and complementary in vivo and in vitro assays for endocytic clathrin coated vesicle (CCV) formation and will use these to probe the fundamental mechanisms underlying CME. The in vivo assays utilize total internal reflection fluorescence microscopy coupled with novel tracking software and statistical analyses that allow us to comprehensively and quantitatively analyze clathrin coated pit (CCP) dynamics and the kinetics of CCV formation in living cells. The in vitro assay utilizes supported lipid bilayers with excess membrane reservoir or 'SUPER' templates, which provide a robust and facile assay system for vesicle formation that is amenable to both biochemical analysis and real-time imaging. Using these assays, we will pursue three major aims: 1) To quantitatively define factors that regulate clathrin coated pit initiation and maturation in vivo, including cargo, PI4,52, v-SNAREs and AP2 interactions; 2) To define the role of SH3 domain-containing dynamin-1 effectors as regulators of dynamin function in CCV formation in vivo and in vitro; and 3) To reconstitute CCV formation from SUPER templates using purified protein components. By defining the minimum machinery required for CCV formation and establishing a robust, quantitative and physiologically relevant assay for measuring this process we will have established the means to fully understand the mechanistic underpinnings of clathrin-mediated endocytosis. )